Background. Synapsin is a crucial regulator of neurotransmission and allows synapses to maintain a large reserve pool of synaptic vesicles. Human mutations in synapsin genes are linked to epilepsy and autism. How synapsin function is regulated to allow replenishment of synaptic vesicles and sustain neurotransmission is largely unknown. Liquid-liquid phase separation is an increasingly recognized mechanism for membrane-less subcellular compartmentalization, in which a distinct liquid phase forms in the cytosol due to weak multimeric interactions between proteins, or between RNA and proteins [1], [2]. In support of the liquid phase model for SV clustering, in vitrostudies have shown that pure synapsin, or its isolated intrinsically disordered region (IDR), can form droplets in aqueous solution [3]. Droplet formation was enhanced by adding synapsin-binding SH3 domains and disrupted by CaMKII phosphorylation. Moreover, small liposomes could be captured in synapsin droplets [3]. At present, this model of SV clustering has not been tested in vivo. Moreover, with regard to liquid-liquid phase separation, in vitro results are not easily transferred to a cellular context [4].
Aims of the study: To examine whether the requirements for synapsin droplet formation in vitro pertain to SVs clusters at living central synapse.
Methods: Experiments were performed in river lampreys (Lampetra fluviatilis). These animals have synapses that are established by reticulospinal axons on dendrites of spinal neurons and characterized by large synaptic vesicle clusters, which accumulate on the surface of the axons. Isolated preparations of the lamprey spinal cord were prepared and giant axons microinjected with Alexa488-tagged reagents as earlier described [5]. In the present experiments we maintained synapses at rest. Preparations were fixed in glutaraldehyde and embedded in Durcupan ACM[5]. Serial ultrathin sections were cut from the specimens, stained and examined in a Tecnai 12 electron microscope. For quantitative analysis, the number of synaptic vesicles (SVs) was determined from middle sections of at least five serially cut synapses. The values for the numbers of SVs were normalized to the length of the active zone. Statistical analysis of two groups was evaluated using Student’s t-tests. To evaluate the differences between more than two groups one-way analysis of variance (ANOVA) was used, either with Tukey’s post-test comparing every mean with every other mean or Bonferroni multiple comparison test comparing selected pairs of means. Statistical analysis was performed using GraphPad Prism 6.0 (GraphStat Software, San Diego, CA, USA).
Results: Axons were maintained at rest to examine if reagents introduced into the cytosol would enter a putative liquid phase to disrupt critical protein-protein interactions. We found that compounds that perturb the intrinsically disordered region of synapsin, which is critical for liquid phase organization in vitro, such as SH3A domain of Intersectin 1 and antibodies against IDR region of synapsin, cause dispersion of synaptic vesicles from resting clusters. Reagents that perturb SH3 domain interactions with synapsin were found ineffective at rest.
Conclusions: Our results indicate that synaptic vesicles at a living central synapse are organized as a distinct liquid phase maintained by interactions via the intrinsically disordered region of synapsin.
This study was supported by the Swedish Research Council (proj. 1501), Parkinsonfonden, Hjärnfonden and RSF grant, project 16-15-10273.

References
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[4] Alberti, S., Gladfelter, A., and Mittag, T. (2019). Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates. Cell 176, 419-434.
[5] Wen, P.J., Grenklo, S., Arpino, G., Tan, X., Liao, H.S., Heureaux, J., Peng, S.Y., Chiang, H.C., Hamid, E., Zhao, W.D., Shin, W., Näreoja, T., Evergren, E., Jin, Y., Karlsson, R., Ebert, S.N., Jin, A., Liu, A.P., Shupliakov, O., Wu, L.G. (2016) Actin dynamics provides membrane tension to merge fusing vesicles into the plasma membrane. Nature Commun. 7:12604.